Current cancer therapy is based on radiotherapy, surgery, sometimes very crippling and/or the use of anti-cancer drugs which block mitoses and which can be very aggressive, which sometimes limits their use. There is currently no universal therapy against this pathology. The role of angiogenesis in tumour growth has been the subject of intensive research and it is now accepted by all the scientific community that tumour growth cannot take place without angiogenesis. This mechanism is defined as a dynamic process induced by a certain number of angiogenic factors of which the principal ones are: vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) or heparin affin regulatory peptide (HARP) which in several cases are overexpressed by the tumours themselves. Since a tumour cannot develop without neo-vascularisation, the suppression or the inhibition of the angiogenic factors must lead to a regression of the tumour growth regardless of the type of tumour. Nowadays in the treatment of cancer or of proliferative diseases several companies are developing an anti-angiogenesis strategy based for example either on inhibitors of angiogenic factors (or of receptors thereof when these are identified), or by inducing vascular micro-thromboses using the endothelial cell as anchorage for catalysts of the thrombosis, or even by using peptide agents which inhibit angiogenesis by mechanisms as yet unidentified. These approaches have not yet produced clear results and it appears that the inhibitors based on blocking one single angiogenesis pathway induce aggravating rebound effects. These results are currently leading these companies to propose cocktails of inhibitors which make it possible to hope for radical simultaneous destruction of the vessels and tumour cells.
The angiogenic role of the HARP factor has been demonstrated through a number of experiments conducted in vitro and in vivo (Papadimitriou et al 2001; Papadimitriou et al, 2000). Thus in an in vitro model in which endothelial cells are seeded on a collagen gel it has been shown that HARP is capable of inducing the formation of pseudo-capillaries, thus mimicking the first stages of angiogenesis, that is to say the activation of the endothelial cells and their migration through a partially destroyed extracellular matrix. Reinforcing this observation, a synthetic peptide of 43 amino acids corresponding to a part of the C-terminal domain of HARP is capable of stimulating the secretion of the plasminogen activator by bovine aortic endothelial cells (ABAE) and inhibiting the secretion of its inhibitor PAI-1 (Kojima et al, 1995a; Kojima et al, 1995b). This activator induces the cleavage of the plasminogen in plasmin, a protease which plays a key role in the degradation of the extracellular matrix. HARP shares this characteristic with another protein which binds to heparin (Matsubara et al, 1990): the protein midkine (MK) which exhibits 50% homology of amino acid sequence with which it constitutes a new family of HBGF (Tsutsui et al, 1991).
The role played by HARP in tumoral angiogenesis has also been underlined by Roy Bicknell's team. Thus the overexpression of HARP in the MCF7 mammary carcinoma cells injected into “nude” mice results in an increase in the size of the tumour relative to those obtained without overexpression. The increase in the size of the tumour is linked to the vascular density and to a multiplication of the endothelial cells (Choudhuri et al, 1997). The proliferation of the endothelial cells is another key stage in angiogenesis in which HARP could play a crucial role. In fact, since 1991 it has been shown that HARP stimulated the proliferation of the endothelial cells in vitro (Courty et al, 1991). This mitogenic activity could be demonstrated in other models: HARP stimulates in vitro the formation of colonies of non-tumorigenic epithelial cells SW13 (Fang et al, 1992) in soft agar and the overexpression of its cDNA in NIH 3T3 cells (Chauhan et al, 1993) or SW-13 cells (Fang et al, 1992) induces the formation of a tumour in the “nude” mouse. This mitogenic role is reinforced by the expression of the molecule in numerous cancers. By the use of RNAse protection tests and/or the Northern blot technique, HARP mRNA was detected in the cell lines coming from breast cancer (T47 Dco, MDA-MB231, MDA-MB361, Hs-578T) (Fang et al, 1992), ovarian cancer (A1827, PA-1) (Riegel et al, 1994), prostate cancer (PC-3) (Vacherot et al, 1999a) and lung cancer (Jager et al, 1997).
In vivo, in different tissue models the localisation of HARP is in particular associated with the endothelial cells of the blood capillaries. mRNA for HARP protein was demonstrated in the endothelial cells belonging to the blood capillaries of human prostate and mammary glands (Vacherot et al, 1999a; Ledoux et al, 1997). In the rat uterine model there was also demonstrated an increase in the rate of mRNA and of HARP protein associated with the progestative phase of the cycle. This result was confirmed by the injection of progesterone into ovariectomised rats. The overexpression induced by the progesterone is detectable in the capillary endothelial cells of the endometrium (Milhiet et al, 1998). The presence of the HARP protein on the surface of the endothelial cells has also been demonstrated indirectly during intravenous injections of heparin in humans (Novotny et al, 1993). Recently the Deuel group demonstrated an increase in the expression of the mRNA of HARP in the microvessels in development after a cerebral ischaemia in the rat (Yeh et al, 1998). The growth factors HARP and midkine (MK) are molecules which exhibit 50% homology and possess mitogenic properties on epithelial, fibroblastic and endothelial cells. The expression of each of these growth factors (mRNA, protein) was demonstrated in human tumours of varied origin (breast, lung, ovary, neuroblastoma, stomach, colon), suggesting their potential role in the course of the tumoral progression. A few clinical studies have evaluated the coexpression of HARP and of MK and in particular the blood level of these angiogenic molecules in patients with tumours. Recently an immunological dosage for these two molecules has been developed based on their affinity for heparin. The sensitivity of the method is 80 pg/ml for HARP and 40 pg/ml for MK (Stoica et al, 2001).
U.S. Pat. No. 5,641,743 and U.S. Pat. No. 6,103,880 describe in a general manner the HARP protein and the use of this protein to stimulate angiogenesis.
The Patent Application FR 2 799 465 discloses a fragment of HARP with angiogenic activity.
In fact it has been shown that the angiogenic activity of HARP occurred through a smaller peptide. Thus a consensus sequence of 18 amino acids resulting from the HARP peptide sequence but also found on a large number of angiogenic factors possessed an angiogenic activity by itself.
Like the majority of the growth factors, the mechanism of action of HARP is brought about by an interaction with a membrane receptor with high affinity tyrosine kinase activity (KD=50 pM) known as “anaplastic lymphoma kinase” or ALK (Stoica et al, 2001).
Furthermore, the biological activity of HARP, like the majority of HBGFs, also depends upon its interaction with glycoaminoglycans or GAG, of the heparan or chondroitin sulphate type (Vacherot et al, 1999b).
Thus the mitogenic activity of HARP is potentiated in the presence of heparin, heparan sulphate and chondroitin sulphate of type A and B. Cellular treatment with heparinase abolishes the mitogenic activity of HARP. This activity can then be restored in the presence of heparin. A study relating to the relationships between the structure and the function of HARP has suggested that the C-terminal part of HARP corresponding to the amino acid residues 111-136 (numbering with reference to the form HARP136) is directly implicated in the induction of the mitogenic activity (Bernard-Pierrot et al, 2001) and in its interaction with the receptor ALK. Moreover it has been established that the peptide 111-136 is neither mitogenic n or angiogenic and that the HARP molecule from which the amino acids 111-136 have been deleted specifically inhibits the biological activity of HARP whilst having negative dominant effects with regard to HARP (Bernard-Pierrot et al 2002).